One of the complications of venoarterial extracorporeal membrane oxygenation (VA ECMO), an accepted rescue therapy for term and near-term infants in respiratory failure, is the development of major intracranial lesion along with hypertension. Developmental deficits are also reported in half of the ECMO population. Studies in the newborn lamb model of non-pulsatile VA ECMO have demonstrated that short term exposure to ECMO and hypoxia results in an impaired cerebral blood flow (CBF) autoregulatory response. Loss of CBF autoregulation, i.e. altered vascular reactivity makes the cerebral vascular bed pressure passive. This altered vascular reactivity is very likely to be a major cause of intracranial injury in the ECMO population. Our major goal is to understand the mechanisms underlying hypoxia and ECMO-induced loss of CBF autoregulation. Diffusible endothelial vasoactive factors, produced in response to changes in hemodynamic sheer stress associated with blood flow, modulate vascular tone. The applicant has obtained recent data which suggest that exposure of newborn lambs to 2 hours of VA ECMO or hypoxia decreases the basal production of nitric oxide (NO) in cerebral arteries. The present proposal is to understand the mechanisms for this functional impairment. Aim I is to test the hypothesis that non-pulsatile flow in cerebral arteries impairs basal and stimulated release of NO. We will study the effect of pulsatility during ECMO by using the traditional non-pulsatile pump and also a pulsatile pump. Isolated arteries from the animals will be tested in a pressurized arteriograph to examine the status of various biochemical mechanisms of contraction and dilation, and for the direct measurement of NO released by the endothelium in response to pulsatile flow. We have obtained preliminary data in our animal model that hypoxia followed by reoxygenation selectively impairs cerebrovascular endothelial function as well as CBF autoregulation. Aim 2 is to define and characterize the mechanism of endothelial damage caused by hypoxia by itself and in combination with VA ECMO. Based on recent data obtained by the applicant, Aim 3 is to test the hypothesis that impaired generation of NO after VA ECMO and hypoxia is related to changes in association of Hsp90 and endothelial nitric oxide synthase (eNOS). The co-association of these two proteins will be studied in cerebral arteries obtained from animals exposed to ECMO and hypoxia. Results from these studies will define the roles of pulsatility and hypoxia in the regulation of cerebral arterial function and in the regulation of the activity of eNOS. These studies will also improve our understanding of the underlying mechanisms of ECMO-induced injury of cerebral arterial endothelium, and impairment in CBF autoregulation.